CN106267175B

CN106267175B - Chicken coccidiosis vaccine and application thereof

Info

Publication number: CN106267175B
Application number: CN201610833488.5A
Authority: CN
Inventors: 索勋; 汤新明; 刘贤勇; 索静霞
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2016-09-19
Filing date: 2016-09-19
Publication date: 2020-01-17
Anticipated expiration: 2036-09-19
Also published as: CN106267175A

Abstract

The invention relates to the field of biological immunity, and particularly discloses a coccidiosis vaccine for chickens and application thereof. The transgenic coccidian is taken as a vaccine component, so that the immune effect of the existing coccidiosis vaccine can be further improved, the side effect generated by the immunization of the coccidiosis vaccine is reduced, and the vaccine has good economic benefit and wide application prospect.

Description

Chicken coccidiosis vaccine and application thereof

Technical Field

The invention relates to the field of biological immunity, in particular to a coccidiosis vaccine for chickens.

Background

Coccidiosis seriously harms the health of chicken flocks and restricts the output of meat and egg products, and is one of important epidemic diseases which limit the development of intensive chicken raising industry. The etiology is 7 Eimeria coccidia, which parasitize different parts of the intestinal tract. Prevention and control strategies rely on drug prophylaxis and vaccination. The coccidiosis vaccine taking the live oocysts as the vaccine component has good prevention and control effects, is the only protozoa live vaccine approved to be on the market so far, and is widely applied to the prevention of breeding hens and laying hens. The prevention of the coccidiosis of the broiler chickens is implemented by coccidiosis vaccination taking live oocysts which are controlled or weakened by medicaments as vaccine components. However, the rapid generation of coccidian drug resistance and the difficulty in developing new drugs make the drug prevention and control unable to meet the requirements of modern green culture. Unfortunately, coccidiosis vaccines have limited their widespread use in the control of coccidia in broiler chickens in view of the properties of their own oocyst components.

The immunological and biological characteristics of coccidia determine that the coccidia vaccine taking the coccidia as the vaccine component cannot adapt to the coccidia control of small-day-old (42-day-old or earlier) slaughtering broilers. The immunogenicity of different coccidia species is greatly different, and the strong immunogenic species such as the eimeria maxima can provide good immune protection after one-time immunization; less immunogenic species such as eimeria tenella need to be boosted for 2-3 times, i.e. oocysts in bedding are repeatedly taken to establish a good immune response, which requires 4-5 weeks or even longer, and the weight loss caused by such boosting cannot be compensated for in the young broiler before slaughtering. On the other hand, the strong pathogenic species such as Eimeria necatrix have poor immunogenicity, and the production performance of the species is seriously affected by the damage to chicken flocks in the repeated immunization process, so that the coccidiosis vaccine applied to the broiler chicken does not generally comprise the component. The method is undoubtedly the biggest problem of preventing and controlling the coccidiosis of the broilers, and is a big bottleneck for limiting green and healthy breeding of the broilers.

Disclosure of Invention

In order to solve the problems existing in the existing coccidiosis vaccine immunization, the invention aims to provide a novel chicken coccidiosis vaccine immunization method, which changes the existing coccidiosis vaccine immunization strategy or immunization program, shortens the time for establishing protective force of vaccine immunization, further reduces the negative effect of chicken after taking oocysts discharged after self-vaccination, and simultaneously can provide good immune protection against coccidiosis infection for chicken flocks.

In order to realize the purpose of the invention, the invention provides a chicken coccidiosis vaccine and application thereof.

In a first aspect, in order to make the immune control of coccidiosis safer and more effective, the invention provides an improved chicken coccidiosis vaccine, which comprises an immunogen causing protective immune response, wherein the immunogen is a transgenic coccidian capable of expressing a factor related to the regulation of the host immune response strength.

Wherein, the factors related to the regulation of the host immune response strength comprise cytokines, ligands of immune receptors and insect antigens which can regulate and control the immune response among different insect species.

The cytokine is selected from Th1 type cytokine, preferably IFN-gamma.

The ligand of the immunoreceptor is selected from a synergistic factor of activated T cells, i.e. a second signaling factor, preferably an antibody Fc fragment or CD 40L.

The insect body antigen capable of regulating and controlling immune response among different insect species is selected from antigen factors combined with dendritic cell surface receptors, and is preferably Profilin.

The construction method of the transgenic coccidian comprises the following steps:

1) constructing a coccidian transfection vector containing the cytokine gene, utilizing the coccidian self regulation elements such as a promoter and the like, and utilizing restriction endonuclease linearization after mass preparation;

2) the technique of nuclear transfection mediated by restriction enzymes (transfection instrument: NucleofectorII, AMAXA, program U-033) to transfect linearized vectors into coccidia sporozoites;

3) the transfected sporozoites are inoculated to chicks through cloaca to obtain transgenic coccidian oocysts, and stable genetic groups are obtained through continuous screening and monoclonal sorting.

Optionally, the nucleotide sequences of the chicken IFN-gamma, the chicken IgY Fc, the chicken CD40L and the E.maxima Profilin are shown in SEQ ID NO. 1-4.

In a second aspect, the invention further provides the use of said vaccine.

Preferably, the application is: orally immunizing coccidiosis vaccine for 1 day old chicks; after 1-3 days, the same coccidiosis vaccine is orally immunized again.

The chicks include, but are not limited to, broilers, layers or breeders.

The coccidiosis vaccine may be a commercially available commercial vaccine, and may preferably be a transgenic coccidiosis vaccine as provided hereinafter.

After the immune chicken is immunized for the first time, the time for discharging coccidian oocysts is about 5-14 days. According to the immunization method, after 1-3 days of the first immunization, the second immunization is carried out through oral administration again, so that the second immune response can be carried out before the immune chicken ingests the oocysts discharged by the immune chicken, and the serious damage to the intestinal tract caused by excessive ingestion of the oocysts before the immune protection of the chicken is not established is avoided.

Experimental research shows that if the second immunization is carried out 2 days after the first immunization, the optimal immune protection effect can be achieved, and the specific expression is that oocysts are less discharged after insect attack infection, lesions are lighter, and the weight gain is better.

Further, the immunization dose of the vaccine is 1/2 of the recommended dose of the commercial vaccine. For example, the recommended dose of a chicken coccidiosis tetravalent live vaccine (Fushan Zhengdian Biotechnology limited company, veterinary Sheng word (2008)190462139) is 1100 + -10% (275 + -10% oocyst/species), and the vaccine is applied to the present invention, and is divided into two immunizations, wherein each dose is 550 + -10%.

In order to avoid larger recommended dose difference of the commercial vaccine, the invention further provides a more definite dose standard, namely the doses of the two vaccine immunizations are respectively 100-800 oocysts/species/feather, preferably the doses of the two vaccine immunizations are the same, and preferably 125-175 oocysts/species/feather. When in use, the dosage recommended by the commercial vaccine and the dosage of the invention can be referred at the same time.

The invention has the beneficial effects that:

the invention provides a coccidiosis vaccine, which is characterized in that transgenic coccidiosis expressing relevant factors (including cell factors, ligands of immune receptors and insect antigens capable of regulating and controlling immune response among different insect species) for regulating the immune response strength of a host is constructed by utilizing a transgenic technology. The transgenic coccidian is taken as a vaccine component, so that the immune effect of the existing coccidiosis vaccine can be further improved, the side effect generated by the immunization of the coccidiosis vaccine is reduced, and the vaccine has good economic benefit and wide application prospect.

In addition, the invention also provides an application method of the vaccine, which can effectively avoid the risk that a large amount of oocysts are discharged after primary immunization and are eaten by chicken flocks to cause coccidiosis, improve the uniformity of the coccidiosis vaccine immunization to more than 90 percent, quickly establish immune protection, realize better protection on the re-infection of the coccidiosis, slow down the reduction of the production performance caused by the vaccine immunization and increase the breeding benefit. Meanwhile, the dosage of the anti-coccidium medicine can be reduced or eliminated, and safer and more green poultry meat and egg products are provided. Moreover, the cost of the existing immunization method is saved, and the economic benefit is huge.

Drawings

FIG. 1 shows the immunization strategy of example 1 of the present invention.

FIG. 2 shows experimental groups of Experimental example 1 of the present invention.

FIG. 3 is a comparison of oocyst output in Experimental example 1 of the present invention.

FIG. 4 is a comparison of the mean intestinal lesion scores in Experimental example 1 of the present invention.

FIG. 5 is a comparison of body weights in Experimental example 1 of the present invention.

FIG. 6 is a comparison of immunogenicity and protection of transgenic Eimeria tenella (Trans 1) expressing cytokines (Interferon-gamma, IFN-gamma) constructed according to the present invention with a conventional vaccine component, wild-type coccidia (WT). Wherein: (A) the oocyst discharge dynamic rule of each group of chicken after immunization; (B) oocyst output of the parasites at 14 days post immunization; (C) the weight of the chicken population is increased before and after the insect attack in 14 days after immunization; (D) attack insects 14 days after immunization, and score caecal lesion; (E) oocyst output of the parasites 28 days post-immunization; (F) the weight of the chicken population is increased before and after the insect attack 28 days after immunization; (G) the insects were attacked 28 days after immunization and scored for cecal lesions.

FIG. 7 is a comparison of immunogenicity and protection of transgenic Eimeria tenella (Trans 2) against a conventional vaccine component, wild-type coccidia (WT), for a ligand (antibody Fc fragment) of an immunoreceptor constructed according to the present invention. Wherein: (A) the oocyst discharge dynamic rule of each group of chicken after immunization; (B) oocyst output of the parasites at 14 days post immunization; (C) the weight of the chicken population is increased before and after the insect attack in 14 days after immunization; (D) attack insects 14 days after immunization, and score caecal lesion; (E) oocyst output of the parasites 28 days post-immunization; (F) the weight of the chicken population is increased before and after the insect attack 28 days after immunization; (G) the insects were attacked 28 days after immunization and scored for cecal lesions.

FIG. 8 is a comparison of the immunogenicity and protection of transgenic Eimeria tenella (Trans 3) constructed according to the present invention as a ligand of the immunoreceptor (CD40L) with the conventional vaccine component, wild-type coccidia (WT). Wherein: (A) the oocyst discharge dynamic rule of each group of chicken after immunization; (B) oocyst output of the parasites at 14 days post immunization; (C) the weight of the chicken population is increased before and after the insect attack in 14 days after immunization; (D) attack insects 14 days after immunization, and score caecal lesion; (E) oocyst output of the parasites 28 days post-immunization; (F) the weight of the chicken population is increased before and after the insect attack 28 days after immunization; (G) the insects were attacked 28 days after immunization and scored for cecal lesions.

FIG. 9 is a comparison of immunogenicity and protection of transgenic Eimeria tenella (Trans4) with a traditional vaccine component, wild-type coccidia (WT), for a different species of immunoregulatory antigen (E.maxima Profile) constructed in accordance with the present invention. Wherein: (A) the oocyst discharge dynamic rule of each group of chicken after immunization; (B) oocyst output of the parasites at 14 days post immunization; (C) the weight of the chicken population is increased before and after the insect attack in 14 days after immunization; (D) attack insects 14 days after immunization, and score caecal lesion; (E) oocyst output of the parasites 28 days post-immunization; (F) the weight of the chicken population is increased before and after the insect attack 28 days after immunization; (G) the insects were attacked 28 days after immunization and scored for cecal lesions.

FIG. 10 is a comparison of the immunogenicity and protection of transgenic Eimeria necatrix (Trans5) of the differentially immunomodulatory antigen (E.maxima Profile) between different species constructed in accordance with the invention and a conventional vaccine component, i.e., wild-type coccidia (WT). Wherein: (A) the oocyst discharge dynamic rule of each group of chicken after immunization; (B) oocyst output of the parasites at 14 days post immunization; (C) the weight of the chicken population is increased before and after the insect attack in 14 days after immunization; (D) attack insects 14 days after immunization, and score small intestine pathological changes; (E) oocyst output of the parasites 28 days post-immunization; (F) the weight of the chicken population is increased before and after the insect attack 28 days after immunization; (G) the worms were attacked 28 days after immunization and scored for small bowel disease.

FIG. 11 is a comparison of immunogenicity and protection of transgenic Eimeria acervulina (Trans6) of the differentially immunomodulatory antigen (E.maxima Profile) between different species constructed in accordance with the invention and a conventional vaccine component, i.e., wild-type coccidia (WT). Wherein: (A) the oocyst discharge dynamic rule of each group of chicken after immunization; (B) oocyst output of the parasites at 14 days post immunization; (C) the weight of the chicken population is increased before and after the insect attack in 14 days after immunization; (D) attack worm 14 days after immunization, and score for duodenal lesion; (E) oocyst output of the parasites 28 days post-immunization; (F) the weight of the chicken population is increased before and after the insect attack 28 days after immunization; (G) the worms were attacked 28 days after immunization and scored for duodenal lesions.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The chicken coccidiosis tetravalent live vaccine (hereinafter referred to as coccidian vaccine) described in the following examples was purchased from positive classical biotechnology limited of Foshan.

Example 1

This example illustrates the immunization protocol of the present invention.

As shown in figure 1, 1 day old chicks were immunized by oral coccidiosis vaccine (dose 550 + -10% oocysts/feather) normally fed with feed without coccidiosis drug, freely drunk water, and 2 days after primary immunization (3 days old chicks) for re-immunization with the same dose (550 + -10% oocysts/feather).

Example 2

This example differs from example 1 in that: the secondary immunization time was 1 day after the primary immunization (2 days old chicks).

Example 3

This example differs from example 1 in that: the secondary immunization time was 3 days after the primary immunization (chicks were 4 days old).

Experimental example 1

This example is intended to illustrate the different protective effects of the immunization method of the invention on the re-infection of chickens with coccidia compared to the immunization methods of the prior art.

The test method is as follows:

1. set up the packet, as shown in FIG. 2:

(1) the immune group of the invention: as in example 1. Attack infection is carried out 14 days after immunization (dose is 10000 plus or minus 10% of oocysts/seeds/feather, and total dose is 40000 plus or minus 10% of oocysts and feather).

(2) Prior art immunization groups: the 1-day-old chicks are orally immunized with coccidian vaccines (the dose is 1100 +/-10% of oocysts/feathers), and are normally fed with feeds without anticoccidial drugs and freely drink water. At 2 days after the primary immunization, PBS was used as a control. Attack infection is carried out 14 days after immunization (dose is 10000 plus or minus 10% of oocysts/seeds/feather, and total dose is 40000 plus or minus 10% of oocysts/feather).

(3) Non-immune attack group: the 1-day-old chicks were orally immunized with PBS and normally fed with feed without anticoccidial drugs, and were allowed to drink water freely. At 2 days after the primary immunization, PBS was used as a control. Attack infection is carried out 14 days after immunization (dose is 10000 plus or minus 10% of oocysts/seeds/feather, and total dose is 40000 plus or minus 10% of oocysts/feather).

(4) Non-immune non-pest group: the 1-day-old chicks were orally immunized with PBS and normally fed with feed without anticoccidial drugs, and were allowed to drink water freely. At 2 days after the primary immunization, PBS was used as a control. Oral PBS was used as a control 14 days after immunization.

2. Evaluation index

And (3) counting oocyst discharge after immunization and after attack of the insects, analyzing intestinal lesions and weight changes before immunization, after immunization and after attack of the insects, and analyzing the difference of the protection effects of the immune chicken flocks with different immunization programs.

3. Analysis of results

As shown in fig. 3, the oocyst discharge after immunization and attack by the chicken flock of the immunization group is significantly lower than that of the immunization group in the prior art, which shows that the immunization program of the invention can effectively reduce the oocyst discharge after immunization by the chicken flock vaccine and provide better immune protection, and the discharge of the oocysts after attack by the insect is significantly reduced, which shows that the immunization program of the invention significantly reduces the side effect of vaccine immunization and enhances the protective effect of vaccine immunization.

As shown in figure 4, the intestinal lesion score of the chickens attacked by the pests in the immune group is significantly lower than that of the immune group in the prior art, which indicates that the immune program of the invention can provide better immune protection, significantly reduce the side effect of vaccine immunity and obviously enhance the protective effect of vaccine immunity.

As shown in fig. 5, the body weight gain of the immunized group of chickens after being immunized and attacked by insects is significantly higher than that of the immunized group of the prior art, which indicates that the immunization program of the present invention can effectively reduce the production performance reduction caused by immunization of the vaccine of the chickens, and provide better immune protection, which is shown in that the body weight gain after being attacked by insects is significantly increased, and indicates that the immunization program of the present invention significantly reduces the side effects of vaccine immunization, and the protective effect of enhancing vaccine immunization is significant.

Example 4

This example illustrates the improved coccidiosis vaccine provided by the present invention, the immunogen of which is transgenic Eimeria tenella (Trans 1) expressing cytokines (Interferon- γ, IFN- γ).

The preparation method of the vaccine comprises the following steps:

1) constructing an IFN-gamma expressing coccidian transfection vector by utilizing a sequence shown in SEQ ID NO.1, utilizing coccidian self regulation elements such as a promoter and the like, and utilizing restriction endonuclease linearization after mass preparation;

This example is directed to Eimeria tenella (Eimeria tenella), one of the most serious species of coccidia harming the poultry industry, using the transgenic coccidia listed in this invention expressing cytokines (Interferon-gamma, IFN-gamma), and responding to their immune effects by oocyst expulsion, cecal lesion scoring, and weight change.

200 feather 1 week old SPF chickens were randomly divided into 4 groups: non-immune non-attack group, non-immune attack group, traditional vaccine coccidia component, i.e. wild type coccidia immune group (WT) and transgenic coccidia immune group (Trans 1). Each group of chickens was inoculated with 200. mu.l PBS, 200. mu.l PBS (containing 200WT oocysts) and 200. mu.l PBS (containing 200Trans 1 oocysts) orally, and the chickens were housed in the same condition in flat stock and were fed with water and water freely. On 14 days after immunization, 10000 oocysts/feather of 25 chickens in each group except the group without immunization and attack of insects are orally infected and transferred to a new isolator for feeding, 10 chickens are killed on 6 days after infection, and the oocyst discharge and body weight gain of the chickens are counted. The remaining 25 chickens were treated identically 28 days after immunization. In order to reflect the immune protection effect of the immunogenicity-enhanced coccidiosis vaccine, the oocyst output after immunization and attack, the weight change of chickens before and after immunization and after attack of insect infection and the pathological change degree of caecum are counted in the case, and the details are as follows:

1. the expulsion of oocysts in feces after immunization (fig. 6A) shows that the immunogenicity-enhanced transgenic coccidia immune group is significantly lower than the wild type coccidia immune group, reducing the risk of coccidiosis outbreak after the novel vaccine is immunized. Oocyst shedding in feces after challenge on day 14 of immunization (FIG. 6B), it can be seen that the Trans1 immunization group provided better immune protection and reduced oocyst shedding compared to the wild-type coccidia immunization group. Oocyst shedding in feces after challenge on day 28 of immunization (FIG. 6E) it can be seen that both the Trans1 immunization group and the wild type coccidia immunization group provided enhanced immune protection, but the Trans1 immunization group was superior to the wild type coccidia immunization group.

2. Body weight gain of chicken flocks before and after the attack on day 14 of immunization (fig. 6C), it can be seen that the Trans1 immunized group provided better immune protection with less effect on body weight gain than the wild type coccidia immunized group. The body weight gain of the chicken flocks before and after the attack on day 28 of immunization (FIG. 6F) can be seen, and the immune protection provided by the Trans1 immune group and the wild type coccidia immune group is enhanced, but the Trans1 immune group is better than the wild type coccidia immune group.

3. Caecal lesions in the flock 6 days after challenge on day 14 of immunization (fig. 6D), it can be seen that the Trans1 immunization group provided better immune protection and less lesions than the wild type coccidia immunization group. Caecal lesions of the chicken flocks 6 days after the attack on the 28 th day of immunization (fig. 6G), it can be seen that the immune protection provided by the Trans1 immunization group and the wild type coccidia immunization group is enhanced, but the Trans1 immunization group is superior to the wild type coccidia immunization group.

Example 5

This example illustrates the improved coccidiosis vaccines provided by the present invention, in which the immunogen is a transgenic Eimeria tenella (Trans 2) expressing the ligand of the immunoreceptor (antibody Fc fragment)

The preparation method of the vaccine is different from that of the vaccine in example 4 in that IFN-gamma gene is replaced by antibody Fc fragment gene (shown in SEQ ID NO. 2).

This example is directed to one of the most serious coccidia species in poultry farming, Eimeria tenella (Eimeria tenella), using a transgenic coccidia listed in the present invention expressing a ligand of an immunoreceptor (antibody Fc fragment), and responding to its immune effect by oocyst expulsion, cecal lesion scoring, and weight change.

200 feather 1 week old SPF chickens were randomly divided into 4 groups: non-immune non-attack group, non-immune attack group, traditional vaccine coccidia component namely wild type coccidia immune group (WT) and transgenic coccidia immune group (Trans 2). Each group of chickens was inoculated with 200. mu.l PBS, 200. mu.l PBS (containing 200WT oocysts) and 200. mu.l PBS (containing 200Trans 1 oocysts) orally, and the chickens were housed in the same condition in flat stock and were fed with water and water freely. On 14 days after immunization, 10000 oocysts/feather of 25 chickens in each group except the group without immunization and attack of insects are orally infected and transferred to a new isolator for feeding, 10 chickens are killed on 6 days after infection, and the oocyst discharge and body weight gain of the chickens are counted. The remaining 25 chickens were treated identically 28 days after immunization. In order to reflect the immune protection effect of the immunogenicity-enhanced coccidiosis vaccine, the oocyst output after immunization and attack, the weight change of chickens before and after immunization and after attack of insect infection and the pathological change degree of caecum are counted in the case, and the details are as follows:

1. the expulsion of oocysts in feces after immunization (fig. 7A) shows that the immunogenicity-enhanced transgenic coccidia immune group is significantly lower than the wild type coccidia immune group, reducing the risk of coccidiosis outbreak after the novel vaccine is immunized. Oocyst shedding in feces after challenge on day 14 of immunization (fig. 7B), it can be seen that the Trans 2 immunization group provided better immune protection and reduced oocyst shedding compared to the wild-type coccidia immunization group. Oocyst shedding in feces after challenge on day 28 of immunization (FIG. 7E) it can be seen that both the Trans 2 immunization group and the wild type coccidia immunization group provided enhanced immune protection, but the Trans 2 immunization group was superior to the wild type coccidia immunization group.

2. Body weight gain of chicken flocks before and after the attack on day 14 of immunization (fig. 7C), it can be seen that the Trans 2 immunization group can provide better immune protection with less influence on body weight gain than the wild type coccidia immunization group. The body weight gain of the chicken flocks before and after the attack on day 28 of immunization (FIG. 7F) can be seen, and the immune protection provided by the Trans 2 immune group and the wild type coccidia immune group is enhanced, but the Trans 2 immune group is better than the wild type coccidia immune group.

3. Caecal lesions in the flock 6 days after challenge on day 14 of immunization (fig. 7D), it can be seen that the Trans 2 immunization group provided better immune protection and less lesions than the wild type coccidia immunization group. Caecal lesions of the chicken flocks 6 days after the attack on the 28 th day of immunization (fig. 7G), it can be seen that the immune protection provided by both the Trans 2 immune group and the wild type coccidia immune group is enhanced, but the Trans 2 immune group is superior to the wild type coccidia immune group.

Example 6

This example illustrates the improved coccidiosis vaccine provided by the present invention, the immunogen of which is a transgenic Eimeria tenella (Trans 3) expressing the ligand for the immune receptor (CD 40L).

The preparation method of the vaccine is different from that of the vaccine in example 4 in that IFN-gamma gene is replaced by chicken CD40L gene (sequence shown in SEQ ID NO. 3).

This example is directed to Eimeria tenella (Eimeria tenella), one of the most serious species of coccidia harming the poultry industry, using a transgenic coccidia listed in the present invention expressing a ligand for immunoreceptor (CD40L), and responding to its immune effects by oocyst expulsion, caecum lesion scoring, and weight change.

200 feather 1 week old SPF chickens were randomly divided into 4 groups: non-immune non-attack group, non-immune attack group, traditional vaccine coccidia component namely wild type coccidia immune group (WT) and transgenic coccidia immune group (Trans 3). Each group of chickens was inoculated with 200. mu.l PBS, 200. mu.l PBS (containing 200WT oocysts) and 200. mu.l PBS (containing 200Trans 1 oocysts) orally, and the chickens were housed in the same condition in flat stock and were fed with water and water freely. On 14 days after immunization, 10000 oocysts/feather of 25 chickens in each group except the group without immunization and attack of insects are orally infected and transferred to a new isolator for feeding, 10 chickens are killed on 6 days after infection, and the oocyst discharge and body weight gain of the chickens are counted. The remaining 25 chickens were treated identically 28 days after immunization. In order to reflect the immune protection effect of the immunogenicity-enhanced coccidiosis vaccine, the oocyst output after immunization and attack, the weight change of chickens before and after immunization and after attack of insect infection and the pathological change degree of caecum are counted in the case, and the details are as follows:

1. the expulsion of oocysts in feces after immunization (fig. 8A) shows that the immunogenicity-enhanced transgenic coccidia immune group is significantly lower than the wild type coccidia immune group, reducing the risk of coccidiosis outbreak after the novel vaccine is immunized. Oocyst shedding in feces after challenge on day 14 of immunization (fig. 8B), it can be seen that the Trans 3 immunization group provided better immune protection and reduced oocyst shedding compared to the wild-type coccidia immunization group. Oocyst shedding in feces after challenge on day 28 of immunization (FIG. 8E) it can be seen that both the Trans 3 immunization group and the wild type coccidia immunization group provided enhanced immune protection, but the Trans 3 immunization group was superior to the wild type coccidia immunization group.

2. Body weight gain of chicken flocks before and after the attack on day 14 of immunization (fig. 8C), it can be seen that the Trans 3 immunization group can provide better immune protection with less influence on body weight gain than the wild type coccidia immunization group. The body weight gain of the chicken flocks before and after the attack on day 28 of immunization (FIG. 8F) can be seen, and the immune protection provided by the Trans 3 immune group and the wild type coccidia immune group is enhanced, but the Trans 3 immune group is better than the wild type coccidia immune group.

3. Caecal lesions in the flock 6 days after challenge on day 14 of immunization (fig. 8D), it can be seen that the Trans 3 immunization group provided better immune protection and less lesions than the wild type coccidia immunization group. Caecal lesions of the chicken flocks 6 days after the attack on the 28 th day of immunization (fig. 8G), it can be seen that the immune protection provided by both the Trans 3 immune group and the wild type coccidia immune group is enhanced, but the Trans 3 immune group is superior to the wild type coccidia immune group.

Example 7

This example illustrates the improved coccidiosis vaccine provided by the present invention, the immunogen being a transgenic Eimeria tenella (Trans4) expressing an immunoregulatory antigen that is differential between different species (Eimeria maxima Profile).

The preparation method of the vaccine is different from that of the vaccine in example 4 in that the IFN-gamma gene is replaced by Eimeria maxima Profile gene (shown in SEQ ID NO. 4).

In this example, the transgenic coccidia listed in the present invention expressing immunoregulatory antigen (E.maxima Profilin) which is different among different species is used to target Eimeria tenella (Eimeria tenella), which is one of the most serious coccidia species harming poultry industry, and the immune effect is reflected by oocyst excretion, caecum lesion score and weight change.

200 feather 1 week old SPF chickens were randomly divided into 4 groups: non-immune non-attacking group, non-immune attacking group, traditional vaccine coccidia component, i.e. wild type coccidia immune group (WT) and transgenic coccidia immune group (Trans 4). Each group of chickens was inoculated with 200. mu.l PBS, 200. mu.l PBS (containing 200WT oocysts) and 200. mu.l PBS (containing 200Trans 1 oocysts) orally, and the chickens were housed in the same condition in flat stock and were fed with water and water freely. On 14 days after immunization, 10000 oocysts/feather of 25 chickens in each group except the group without immunization and attack of insects are orally infected and transferred to a new isolator for feeding, 10 chickens are killed on 6 days after infection, and the oocyst discharge and body weight gain of the chickens are counted. The remaining 25 chickens were treated identically 28 days after immunization. In order to reflect the immune protection effect of the immunogenicity-enhanced coccidiosis vaccine, the oocyst output after immunization and attack, the weight change of chickens before and after immunization and after attack of insect infection and the pathological change degree of caecum are counted in the case, and the details are as follows:

1. the expulsion of oocysts in feces after immunization (fig. 9A) shows that the immunogenicity-enhanced transgenic coccidia immune group is significantly lower than the wild type coccidia immune group, reducing the risk of coccidiosis outbreak after the novel vaccine is immunized. Oocyst shedding in feces after challenge on day 14 of immunization (fig. 9B), it can be seen that the Trans4 immunization group provided better immune protection and reduced oocyst shedding compared to the wild-type coccidia immunization group. Oocyst shedding in feces after challenge on day 28 of immunization (FIG. 9E) it can be seen that both the Trans4 immunization group and the wild type coccidia immunization group provided enhanced immune protection, but the Trans4 immunization group was superior to the wild type coccidia immunization group.

2. Body weight gain of chicken flocks before and after the attack on day 14 of immunization (fig. 9C), it can be seen that the Trans4 immunization group can provide better immune protection with less influence on body weight gain than the wild type coccidia immunization group. The body weight gain of the chicken flocks before and after the attack on day 28 of immunization (FIG. 9F) can be seen, and the immune protection provided by the Trans4 immune group and the wild type coccidia immune group is enhanced, but the Trans4 immune group is better than the wild type coccidia immune group.

3. Caecal lesions in the flock 6 days after challenge on day 14 of immunization (fig. 9D), it can be seen that the Trans4 immunization group provided better immune protection and less lesions than the wild type coccidia immunization group. Caecal lesions in the chicken flocks 6 days after the attack on the 28 th day of immunization (fig. 9G), it can be seen that the immune protection provided by both the Trans4 immune group and the wild type coccidia immune group is enhanced, but the Trans4 immune group is superior to the wild type coccidia immune group.

Example 8

This example illustrates the improved coccidiosis vaccine provided by the present invention, the immunogen being a transgenic Eimeria necatrix (Trans5) expressing an immunoregulatory antigen that is differential between different species (Eimeria maxima Profile).

In this example, another coccidia which is the most serious in poultry industry, Eimeria necatrix (Eimeria necatrix) is used as a target, and the transgenic coccidia listed in the invention expressing the immunoregulatory antigen (Eimeria maxima Profilin) which is different among different species is used, and the immune effect is reflected by oocyst excretion, caecum lesion score and weight change.

200 feather 1 week old SPF chickens were randomly divided into 4 groups: non-immune non-attacking group, non-immune attacking group, traditional vaccine coccidia component, i.e. wild type coccidia immune group (WT) and transgenic coccidia immune group (Trans 5). Each group of chickens was inoculated with 200. mu.l PBS, 200. mu.l PBS (containing 200WT oocysts) and 200. mu.l PBS (containing 200Trans 1 oocysts) orally, and the chickens were housed in the same condition in flat stock and were fed with water and water freely. On 14 days after immunization, 10000 oocysts/feather of 25 chickens in each group except the group without immunization and attack of insects are orally infected and transferred to a new isolator for feeding, 10 chickens are killed on 6 days after infection, and the oocyst discharge and body weight gain of the chickens are counted. The remaining 25 chickens were treated identically 28 days after immunization. In order to reflect the immune protection effect of the immunogenicity-enhanced coccidiosis vaccine, the output of oocysts after immunization and attack of insects, the weight change of chicken flocks before and after immunization and after attack of insects and the pathological change degree of small intestines are counted, and the details are as follows:

1. the expulsion of oocysts in feces after immunization (fig. 10A) shows that the immunogenicity-enhanced transgenic coccidia immunized group is significantly lower than the wild type coccidia immunized group, reducing the risk of coccidiosis outbreak after immunization with this new type of vaccine. Oocyst shedding in feces after challenge on day 14 of immunization (FIG. 10B), it can be seen that the Trans5 immunization group provided better immune protection and reduced oocyst shedding compared to the wild-type coccidia immunization group. Oocyst shedding in feces after challenge on day 28 of immunization (FIG. 10E) it can be seen that both the Trans5 immunization group and the wild type coccidia immunization group provided enhanced immune protection, but the Trans5 immunization group was superior to the wild type coccidia immunization group.

2. Body weight gain of chicken flocks before and after the attack on day 14 of immunization (fig. 10C), it can be seen that the Trans5 immunization group can provide better immune protection with less influence on body weight gain than the wild type coccidia immunization group. The body weight gain of the chicken flocks before and after the attack on day 28 of immunization (FIG. 10F) can be seen, and the immune protection provided by the Trans5 immune group and the wild type coccidia immune group is enhanced, but the Trans5 immune group is better than the wild type coccidia immune group.

3. Small intestinal lesions in the flock 6 days after challenge on day 14 of immunization (fig. 10D), it can be seen that the Trans5 immunization group provided better immune protection and less lesions than the wild type coccidia immunization group. Small intestinal lesions of the chicken flocks 6 days after the attack on the 28 th day of immunization (FIG. 10G) can be seen, the immune protection provided by the Trans5 immune group and the wild type coccidia immune group is enhanced, but the Trans5 immune group is better than the wild type coccidia immune group.

Example 9

This example illustrates the improved coccidiosis vaccine provided by the present invention, the immunogen being a transgenic Eimeria acervulina (Trans6) expressing an immunoregulatory antigen that is differential between different species (Eimeria maxima Profile).

In this example, another coccidia which is the most serious in poultry industry, eimeria acervulina, was used as the target, and the immune effect was reflected by oocyst excretion, caecum lesion score and weight change by the transgenic coccidia of the present invention expressing immunoregulatory antigen (eimeria maxima) that is different among different species.

200 feather 1 week old SPF chickens were randomly divided into 4 groups: non-immune non-attacking group, non-immune attacking group, traditional vaccine coccidia component, i.e. wild type coccidia immune group (WT) and transgenic coccidia immune group (Trans 6). Each group of chickens was inoculated with 200. mu.l PBS, 200. mu.l PBS (containing 200WT oocysts) and 200. mu.l PBS (containing 200Trans 1 oocysts) orally, and the chickens were housed in the same condition in flat stock and were fed with water and water freely. On 14 days after immunization, 10000 oocysts/feather of 25 chickens in each group except the group without immunization and attack of insects are orally infected and transferred to a new isolator for feeding, 10 chickens are killed on 5 days after infection, and the oocyst discharge and body weight gain of the chickens are counted. The remaining 25 chickens were treated identically 28 days after immunization. In order to reflect the immune protection effect of the immunogenicity-enhanced coccidiosis vaccine, the output of oocysts after immunization and attack of insects, the weight change of chicken flocks before and after immunization and after attack of insects and the pathological change degree of small intestines are counted, and the details are as follows:

1. the expulsion of oocysts in feces after immunization (fig. 11A) shows that the immunogenicity-enhanced transgenic coccidia immunized group is significantly lower than the wild type coccidia immunized group, reducing the risk of coccidiosis outbreak after immunization with this new type of vaccine. Oocyst shedding in feces after challenge on day 14 of immunization (FIG. 11B), it can be seen that the Trans6 immunization group provided better immune protection and reduced oocyst shedding compared to the wild-type coccidia immunization group. Oocyst shedding in feces after challenge on day 28 of immunization (FIG. 11E) it can be seen that both the Trans6 immunization group and the wild type coccidia immunization group provided enhanced immune protection, but the Trans6 immunization group was superior to the wild type coccidia immunization group.

2. Body weight gain of chicken flocks before and after the attack on day 14 of immunization (fig. 11C), it can be seen that the Trans6 immunization group can provide better immune protection with less influence on body weight gain than the wild type coccidia immunization group. The body weight gain of the chicken flocks before and after the attack on day 28 of immunization (FIG. 11F) can be seen, and the immune protection provided by the Trans6 immune group and the wild type coccidia immune group is enhanced, but the Trans6 immune group is better than the wild type coccidia immune group.

3. Duodenopathy in chickens 5 days after challenge on day 14 of immunization (fig. 11D), it can be seen that the Trans6 immunization group provided better immune protection and less lesions than the wild type coccidia immunization group. Duodenopathy of chicken flocks 5 days after the attack on day 28 of immunization (fig. 11G), it can be seen that immune protection provided by both the Trans6 immune group and the wild type coccidia immune group was enhanced, but the Trans6 immune group was superior to the wild type coccidia immune group.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A coccidiosis chicken vaccine, which is characterized in that the vaccine comprises an immunogen for inducing a protective immune response, wherein the immunogen is a transgenic coccidia capable of expressing a factor related to the regulation of the intensity of the host immune response; the factors related to the regulation of the host immune response strength comprise cell factors, ligands of immune receptors and insect body antigens which can regulate and control the immune response among different insect species; the cell factor is selected from Th1 cell factor; the ligands of the immunoreceptors are selected from synergistic factors of activated T cells; the insect body antigen capable of regulating immune response between different insect species is selected from antigen factors combined with dendritic cell surface receptors.

2. The use of the vaccine of claim 1 in the preparation of a medicament for the treatment of coccidiosis in chickens.